BIOTECHNOLOGY
Kavery Kandasamy
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D. MOLECULAR GENETICS (12 U)
D1. analyze some of the social, ethical, and legal issues
associated with genetic research and biotechnology
D3. demonstrated an understanding of concepts related to
molecular genetics, and how genetic modification is
applied in industry and agriculture
Curriculum Expectations
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• Demonstrate an understanding of genetics manipulation,
including the processes of recombinant DNA technology,
DNA sequencing, and the polymerase chain reaction
• Gain an understanding of the industrial, medical, and
agricultural applications of recombinant DNA technology
• Describe the functions of the cell components used in
genetic engineering, such as restriction enzymes,
methylases, and plasmids
Learning Goals
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1. Introduction
2. History
3. Application
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Bioremediation
Food processing
Forensics
Medicine
4. Tools
5. Controversial Topics
AGENDA
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• Biotechnology- is the use of living organisms, or
substances from living organisms, to develop an
agricultural, medicinal, or environmental product or
process, that would otherwise not be found in a single
organism.
• Recombinant DNA- DNA molecules formed by bringing
together genetic material from multiple organisms that
would otherwise not be found in biological organisms.
INTRODUCTION
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Selective Breeding
• Has been occurring since 2000 years ago, where humans
have been manipulating the phenotype of offspring by
selecting animals and plants for their preferred traits
• Gregor Mendel’s study of Monohybrid and Dihybrid
crossings of pea plants
• Charles Darwin’s study of evolution and natural selection
HISTORY
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Alcohol Fermentation
• Dates to at least tens of
thousands of years ago
• Anaerobic process where
yeast consumes the sugars
and produce ethanol and
carbon dioxide as waste
products
HISTORY
Bread Baking
• The yeast consumes
the sugars in the
dough and produce
ethanol and carbon
dioxide
• The carbon dioxide
creates bubbles in
the dough and the
ethanol is
evaporated in the 7
heating process
BIOREMIDIATION
• The use of organisms to remove or neutralize pollutants
from a contaminated site into less toxic or non toxic
substances
• E.g. Oil degrading microbes
• Research is being conducted
on speeding up this process.
Genetically modifying the
microbes to stimulate its
metabolic capabilities and
optimize growth rates
APPLICATIONS
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BIOREMIDIATION
• Pseudomonas- the oil eater bacterial genus
• There are four different species in the genus, each using a
different component of oil as food source
• In 1975, Dr. Chakrabarty and his team inserted
plasmids from all four species of the oil eaters and put
them into a single microbe. While these plasmids
would usually not operate together in the same cell,
exposing the cell to ultraviolet light caused the
plasmids to join into one.
• Diamond v. Chakrabarty- the case that led to the
first patent of a genetically modified micro-organism
in the U.S.
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AGRICULTURE
• Genetically Modified Foods- are hardier crops that have
been genetically engineered to possess characteristics, that
would otherwise not be found naturally
• In 1981, Eugene Nestor and Mary Dell Chilton used Ti
plasmid from soil bacteria to create transgenic plants
• The bacteria are attracted to chemicals
released by wound and can enter plants
through natural transformation
• The foreign gene is inserted in the T region
and introduced into the plant cell
• Can only infect dicotyledon naturally
(beans, peas and potatoes)
APPLICATIONS
Crown gall
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AGRICULTURE
• Golden Rice
• Rice that contained beta-carotene, pre-cursor
for vitamin A
• Flavr Savr tomato
• Reduced expression of polygalacturonase
(enzyme responsible for ripening and aging of
fruits)
• Bt Corn
• Natural herbicide found in bacteria Bacillus
thuringiensis
• Devastated monarch butterfly population
Activity- Making decisions About DNA Technology:
Golden Rice
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FORENSICS
• Video: DNA- Fingerprinting
Couple A are parents of Baby 3
Couple B are parents of Baby 1
Couple C are parents of Baby 2
APPLICATIONS
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MEDICINE
• Genetic Screening- detection of
mutations known to be associated with
genetic disorders before they manifest
in an individual
• Genetic disorders in the human fetus can be detected
using genetic screening of embryonic cells in the
amniotic fluid
• Genetic counselling- offered to family with the history of
genetic disorders who are at risk of passing mutations to their
off-spring
APPLICATIONS
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MEDICINE
• Gene Therapy- genetically healthy cells are inserted
into the body to counteract disease such as cystic
fibrosis
• Ongoing research for chronic pain
• Antinociceptive transmitters inhibit sensation of pain
• If a therapeutic gene is inserted into the adrenal gland cells to
express these transmitters, more of these molecules will be
made, thus minimizing pain.
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Restriction Endonucleases (RE) – commonly known as
restriction enzymes that cleave double-stranded DNA into
fragments at specific sequences called recognition site.
• Recognition sites
• Complementary palindromic sequence (same sequence when
read in the 5’ to 3’ end)
TOOLS
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Recognition sites
• Sticky ends- fragment end of a DNA molecule with
short single-stranded overhangs
• Blunt ends- fragment ends of a DNA molecule that are
fully base paired
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Methylases- enzymes that add a methyl group to one of the
nucleotides found in a restriction enzyme’s recognition site
• foreign DNA is not methylated and is therefore cleaved
• Plays an important role in protecting a gene fragment from
being cleaved at the wrong location (protects bacterium’s
own DNA)
• Found both in prokaryotes and eukaryotes
TOOLS
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DNA Ligase- enzyme used for joining the strands of DNA
together
• DNA ligase kicks out a water molecule and reforms the
phosphodiester bond of the backbone of the DNA
TOOLS
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• The gene fragment needs
to produce a useful protein
and cellular machinery is
required
• Plasmids- small, circular,
double stranded DNA
molecules, independent of
the chromosomal DNA
that can be replicated and
expressed
PLASMIDS
• Useful for the bacterium
to carry genes that express
proteins for antibiotic
resistance
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• Plasmids contain copy
number- the higher the
copy number, the higher
the number of plasmids
• Multiple cloning siteregion in plasmid that
has been engineered to
contain recognition sites
of a number of RE
PLASMIDS
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• Plasmid and the foreign gene has been excised using the
same RE, producing the same sticky-ends
Cloning
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• When placed together, in
the same solution, the
sticky fragments will
anneal
• DNA Ligase re-forms the
phosphodiester bonds and
once again creates a
circular piece of DNA
• Recombinant DNA- a
combination of the original
plasmid DNA and foreign
DNA
• The plasmid is
introduced into a
bacterial cell to
replicate and form
many copies
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• Plasmids can be used as vectors to carry a desired gene in to a
host cell
• Transformation- the introduction of DNA from another source
(can be natural or chemically induced)
• Bacterial cells are suspended in CaCl solution at 0˚C
• Calcium ions neutralize the negatively charged phosphate
group on the plasmid DNA and on the phospholipids found in
the cell membrane, reducing the repelling of like charges
• A sudden increase in
temperature creates a draft
which sweeps the plasmid
into the bacterial cell
through pores on the
membrane
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Selective Plating
• Used to isolate cells with recombinant DNA
• Vectors also carry antibiotic resistance gene
Successfully
transformed
bacteria will
be able to
grow on media
containing the
antibiotic
Cells that
were not
transformed
will be
eliminated by
the antibiotic
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• How can you check if the foreign gene exists in the
transformed bacteria?
activity
• Individual, transformed colonies are proliferated to obtain
plasmid DNA
• Digested with RE to release cloned fragment
• Gel- Electrophoresis is used to observe for a pattern of
bands
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• Used to separate the fragments of interest from unwanted
fragments
• Based on the chemical and physical properties of DNA
• DNA is negatively charged
• The molar mass of each nucleotide is relatively consistent
• Each nucleotide has the same charge to mass ratio
• The only difference between the two fragments of DNA
that are of differing length is the number of nucleotides
• DNA fragments are separated according to their size
GEL- ELECTROPHORESIS
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• DNA that is digested using RE will be cleaved into
fragments of different lengths
• The shorter the fragment, the faster it will travel as it
navigates easily through the pores in the gel
• Kinesthetic activity
VS.
Retrieved from: http://www.dcclubbing.com/style/obnoxious-things-people-do-in-nightclubs
/
Retrieved from: http://www.dreamstime.com/royalty-free-stockphoto-cute-couple-holding-hands-image22556395
GEL- ELECTROPHORESIS
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• The gel is made up of agarose
(naturally found in seaweed) or
polyacrylamide (artificial)
• DNA samples are placed in a well
(depression in the gel) with a loading
dye and glycerol
• The gel sits amongst an electrolytic
solution called the buffer that will
convey the current through the gel
Retrieved from: http://www.britannica.com/EBchecked/media/40224/In-gelelectrophoresis-an-electric-field-is-applied-to-a
GEL- ELECTROPHORESIS
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• Using direct current, a negative
charge is placed on the side
containing the sample and a positive
charge on the opposite side
• DNA solution will migrate towards
the positively charged electrode
• The migration will allow for the
separation of the DNA fragments of
different size
Retrieved from: http://www.britannica.com/EBchecked/media/40224/In-gelelectrophoresis-an-electric-field-is-applied-to-a
GEL- ELECTROPHORESIS
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• The gel is stained (Ethidium
Bromide is commonly used)
Worksheet + Smartboard activity
• The stain inserts itself into the
DNA and fluoresces under UV
light
• The size of the fragment is then
determined using a molecular
marker as a standard
• Contains fragments of known size
• Plot log of the known fragment
size vs. migrated distance
• Used to interpolate the size of
unknown fragments
Retrieved from: http://journal.nzma.org.nz/journal/120-1265/2817/
GEL- ELECTROPHORESIS
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Polymerase Chain Reaction
• Direct method of making copies of a desired DNA sequence
• Similar process to DNA replication
• Heat (94˚C - 96˚C) is used to separate the two DNA strands
by breaking the hydrogen bonds
PCR
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• DNA primers are used and must be complement to the 3’ to 5’ ends
of the template strands
• The two primers mediate the synthesis of DNA in opposite directions
toward teach other
• Temperature is decreased (50˚C - 65˚C) to allow primers to anneal
• Taq Polymerase (DNA polymerase) builds complementary strands at 72˚C
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• Once complementary strands have been built, the cycle repeats itself.
Each cycle exponentially increases the number of copies of the target
DNA
• The targeted area is not completely isolated in the first few cycles of
DNA replication
• Taq Polymerase adds nucleotides until it reaches the end of the DNA,
which is not necessarily the target area
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PCR Virtual Lab
• By the third cycle, the number of copies of the targeted
strands begins to increase exponentially
• Very effective with only a small amount of DNA
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•
•
•
•
•
BLUE- Adenine
RED- Thymine
GREEN- Guanine
Yellow- Cytosine
White/ Pink- Hydrogen Bonds
PCR paper-clip activity
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5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’
3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’
Primer 1
A-G-A-T-C-G-C-A-A-A-G-C-A-T-T
T-C-T- A-G-C-G-T- T-T- C-G-T-A-A
Primer 2
• Primer 1: 3’ T-A-G 5’
• Primer 2: 5’ G-C-A 3’
PCR paper-clip activity
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3’ T-A-G 5’
5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’
3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’
5’ G-C-A 3’
3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’ New strand
5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’ Original strand
3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’ Original strand
5’ A-G-A-T- C-G-C-A-A-A-G-C-A 3’ New strand
Cycle 1
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5’ G-C-A 3’
3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’ New strand
3’ T-A-G 5’
5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’ Original strand
5’ G-C-A 3’
3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’ Original strand
3’ T-A-G 5’
5’ A-G-A-T- C-G-C-A-A-A-G-C-A 3’ New strand
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5’ A-T-C-G-C-A-A-A-G-C-A 3’
3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’
3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’
5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’
5’ A-G-A-T-C-G-C-A-A-A-G-C-A 3’
3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’
3’ T-A-G-C-G-T- T-T- C-G-T 5’
5’ A-G-A-T- C-G-C-A-A-A-G-C-A 3’
Cycle 2
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5’ G-C-A 3’
3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’
3’ T-A-G 5’
5’ A-T-C-G-C-A-A-A-G-C-A 3’
5’ G-C-A 3’
3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’
3’ T-A-G 5’
5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’ Original strand
5’ G-C-A 3’
3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’ Original strand
3’ T-A-G 5’
5’ A-G-A-T-C-G-C-A-A-A-G-C-A 3’
5’ G-C-A 3’
3’ T-A-G-C-G-T- T-T- C-G-T 5’
3’ T-A-G 5’
5’ A-G-A-T-C-G-C-A-A-A-G-C-A 3’
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5’ A-T-C-G-C-A-A-A-G-C-A 3’
3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’
3’ T-A-G-C-G-T- T-T- C-G-T 5’
5’ A-T-C-G-C-A-A-A-G-C-A 3’
5’ A-T-C-G-C-A-A-A-G-C-A 3’
3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’
3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’
5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’ Original strand
5’ A-G-A-T-C-G-C-A-A-A-G-C-A 3’
3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’ Original strand
3’ T-A-G-C-G-T- T-T- C-G-T
5’ A-G-A-T-C-G-C-A-A-A-G-C-A
3’
5’
5’ A-T-C-G-C-A-A-A-G-C-A 3’
3’ T-A-G-C-G-T- T-T- C-G-T 5’
3’ T-A-G-C-G-T- T-T- C-G-T5’
5’ A-G-A-T-C-G-C-A-A-A-G-C-A 3’
Fragments of interest
Cycle 3
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• Restriction fragment length polymorphism- analysis
that compares the different lengths of DNA fragments
produced by a restriction enzyme digest to determine
genetic differences between individuals
• Polymorphism- any difference in DNA sequence that can be
detected between individuals
• DNA is
digested using
either one or
several RE
RFLP
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The gel appears as a
smear because many
fragments differing only
slightly in size over a
wide range is produced
The gel is subjected to a
chemical that causes
double-stranded DNA to
denature into singlestranded DNA. The
single-stranded DNA is
then transferred to a nylon
membrane with an
electric current (Southern
blotting)
DNA will be transferred
to the positively charged
membrane
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The membrane is
immersed in a solution
containing radioactive
complementary
nucleotide probes that
will attach to
specifically chosen
regions
The probes will bind to
the complementary
bases (hybridization).
The nylon membrane is
placed against X-ray
film
Exposure of the X-ray
film is developed and a
pattern is detected. The
difference can be used to
match DNA from two
sources
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Animal Testing
• Scientists better need to understand how the
body as a whole function under certain
conditions. In order to conduct studies in a
living body, researches must use animals
whose bodies closely resemble those of
humans
• Only four provinces (Alberta, Ontario,
Quebec, and Saskatchewan) have legislation
specific to animals in research
CONTROVERSIAL TOPICS
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Designer Babies
• Case study: A personal-genomics company in California has
been awarded a broad U.S. patent for a technique that could
be used in a fertility clinic to create babies with selected
traits. The patented process from 23andMe, whose main
business is collecting DNA from customers and analyzing it
to provide information about health and ancestry, could be
employed to match the genetic profile of a would-be parent
to that of donor sperm or eggs.
CONTROVERSIAL TOPICS
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